1. Field of the Invention
The present invention relates generally to an assay for a ligand. More particularly, the invention relates to the use of monoclonal antibodies in an assay for L-thyroxine.
2. Description of the Prior Art
Immunologically based diagnostic assays have traditionally used mixtures of antibodies, referred to herein as polyclonal antibodies. Polyclonal antibodies are elaborated in animals by B-lymphocytes in response to the challenge of an antigen, such as a toxin, bacteria, virus or a foreign cell, which invades or is introduced into the animal. Antigens have one or more surface markers, or determinants, which are recognized as foreign by the lymphocytes.
A given lymphocyte recognizes only one determinant of the antigen, and elaborates only a single antibody. The antibody which it elaborates is specific only for an antigen having that determinant. However, most antigens have many determinants, and other lymphocytes will produce antibodies against each of these determinants. In addition, since the number of antigens is unlimited, the individual will likely possess antibodies from previous antigenic challenges. As a result, the individual will possess in its serum a large pool of different, or polyclonal, antibodies.
Known antisera are based on polyclonal antibodies. Even after many isolation and purification steps, polyclonal antibodies are still heterogeneous. Such heterogeneity may limit the specificity of the antiserum and thereby reduce its effectiveness as an immunological reagent, such as a diagnostic reagent.
Monoclonal antibodies are homogeneous and thereby eliminate many of the problems associated with conventional antisera based on polyclonal antibodies. In the preparation of monoclonal antibodies, a mouse is typically injected with the antigen (the immunization step), and, after a period of time, antibody-making lymphocytes are isolated, usually from the spleen. The lymphocytes are fused with myeloma cells to provide fused cells, referred to as hybridomas. The hybridomas are separated from unfused lymphocytes and myeloma cells. Specific hybridomas are isolated and tested to establish that the isolated hybridoma does indeed produce antibody specific for the antigen used in the immunization step. The hybridoma so produced combines the ability of the parent lymphyocyte cell to produce a specific single antibody with the ability of its parent myeloma cell to continually grow and divide, either in vitro as a cell culture or in vivo as a tumor after injection into the peritoneal cavity of an animal.
Monoclonal antibodies possess several advantages over polyclonal antibodies. They are produced by a single hybridoma cell line and are thus absolutely homogeneous. The antigen used in the immunization step does not have to be pure. Monoclonal antibodies are produced by a hybridoma which can grow indefinitely in cell culture or in an animal. Monoclonal antibodies can be obtained in almost unlimited quantity, and the supply is not limited to the lifetime of a producing animal.
Haptens are low molecular weight non-protein substances which are capable of interacting with an antibody, but which are not immunogenic themselves. When haptens are coupled with a protein carrier, they can be made to elicit an immune response by a lymphocyte to produce an antibody. The antibodies thus formed do recognize and react with the hapten in the absence of the protein carrier. Exemplary of haptens are steroids, prostaglandins, thyroxine and various drugs.
Until relatively recently, sensitive and specific methods for measuring the concentration of haptens in serum were not available. In recent years, various immunoassay procedures have been developed. The technique of competitive radioimmunoassay is described in general by the following equation wherein the asterisk represents a radioactive label: ##STR1##
In this procedure, the unlabeled hapten competes with labeled hapten for a limited number of available antibody binding sites, thereby reducing the amount of labeled hapten bound to antibody. The level of radioactivity bound is, therefore, inversely related to the concentration of hapten in the patient sample or standard. After an adequate incubation period, the bound and free fractions are separated and the radioactivity is quantitated.
The amino acid 3,5,3',5'-tetraido-L-thyronine is commonly called thyroxine and is often referred to as T.sub.4. The designation T.sub.4 is understood to mean the L isomer. It is a hormone having as its principal function a stimulating effect on metabolism.
T.sub.4 is the predominant iodothyronine secreted from the thyroid, and the measurement of serum T.sub.4 concentration has become the test commonly employed as an initial procedure in the diagnosis of states of altered thyroid function, such as hyperthyroidism or hypothyroidism. In addition, it is well known that several conditions other than thyroid disease may cause abnormal serum levels of T.sub.4. Among these are pregnancy, estrogenic or androgenic steroids, oral contraceptives, hydantoins and salicylates, stress, hyper- and hypo-proteinemia, and conditions (hereditary or acquired) which cause alterations in serum levels of thyroid binding globulin (TBG) the major serum T.sub.4 transport system.
Early T.sub.4 determinations were indirect measurements of the concentration of protein-bound or butanol-extractable iodine in serum. Later, competitive protein binding (CPB) assays were developed. More recently, radioimmunoassay procedures have been developed which use both polyclonal and monoclonal antibodies.
In general, radioimmunoassay procedures in the art measure counts of radioactivity which are related to the affinity of the antibody for the hapten. Two parameters, B.sub.0 and B, related to the counts of labeled hapten, are used in radioimmunoassay procedures. The B.sub.0 value is the number of counts of labeled hapten bound under specific conditions by a given amount of antibody in the absence of unlabeled hapten. The B value is the number of counts of labeled hapten bound under the same conditions in the presence of unlabeled hapten. In a radioimmunoassay, radioactivity counts are conventionally presented as B/B.sub.0 .times.100, referred to in the art as assay curve parameters, and are compared by plotting curve parameters against hapten concentration.
In order for antibodies to be suitable for use in a radioimmunoassay for T.sub.4, the antibodies must have an affinity for T.sub.4 which is neither too high nor too low. If the affinity of the antibody for T.sub.4 is too low, the assay may not reach equilibrium between bound T.sub.4 and unbound T.sub.4, and any assay design may be invalid. If the affinity of the antibody for T.sub.4 is too high, curve parameters, such as the 100, 90, 50, 10 and 0% ratios of B/B.sub.0 may exhibit excessive deviations from the theoretical straight line plot. A radioimmunoassay for T.sub.4 generally is configured so that the 50% B/B.sub.0 ratio occurs within the accepted "normal" range of serum T.sub.4 concentrations. In the absence of this configuration, determinations of serum T.sub.4 concentrations above and below the normal range may be inaccurate.
Numerous monoclonal antibodies derived from mouse lymphocytes and mouse myeloma cells (mouse-mouse antibodies) have been produced and reported. Representative disclosures are found in the following patents and published applications:
International Application No. PCT/US81/01291, publication No. WO 82/01192 to Trowbridge, and U.S. Pat. Nos. 4,172,124, 4,349,528 and 4,196,265 to Koprowski.
Conventional polyclonal antisera have been used for many years for identifying antigens and haptens. In recent years monoclonal antibodies have been applied to such assays. European Patent Application No. 82302231, publication No. 0064401, to Gillis discloses preparation of antibodies for use in serological detection of the T-cell activator interleukin-2. European Patent Application No. 81303286.9, publication No. 0044722, to Kaplan et al discloses human-human monoclonal antibodies directed to a wide variety of haptens and antigens.
European patent application No. 81105024.4, publication No. 44441, to Molinaro et al. describes mouse-mouse monoclonal antibodies specific to various haptenic drugs, such as gentimicin. Monoclonal antibodies to digoxin were described by Hunter in the J. of Immunology, 129,1165 (1982).
A monoclonal antibody kit for radioimmunoassay of serum T.sub.4 levels is presently marketed by Mallinkrodt, Inc., Immunoassay Systems St. Louis, Mo. under the tradename SPAC.RTM. T.sub.4. Monoclonal antibodies to T.sub.4 are described by Wang et al., in two articles entitled "Monoclonal antibodies to Thyroid Hormones," Monoclonal Antibodies and T-Cell Hybridomas, p. 357, G. Hammerling et al., ed., Elsevier North Holland Biomedical Press, Amsterdam, Netherlands, 1981, and "Monoclonal Antibodies in Clinical Diagnosis," Protides of the Biological Fluids, p. 817, H. Peeters, ed., Pergamon Press, New York, N.Y. 1981. The antibodies described by Wang et al. are hereinafter referred to as Miles antibodies.